Last updated: 2019-07-02

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Knit directory: apaQTL/analysis/

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Unstaged changes:
    Modified:   analysis/NuclearSpecAPAqtl.Rmd
    Modified:   analysis/NuclearSpecIncludeNotTested.Rmd
    Modified:   analysis/Readdistagainstfeatures.Rmd
    Modified:   analysis/overlapapaqtlsandeqtls.Rmd
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    Modified:   code/BothFracDTPlotGeneRegions.sh
    Modified:   code/Snakefile
    Deleted:    code/Upstream10Bases_general.py
    Modified:   code/apaQTLCorrectPvalMakeQQ.R
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    Modified:   code/clusterfiltPAS.json
    Modified:   code/config.yaml
    Modified:   code/environment.yaml
    Modified:   code/makePheno.py
    Deleted:    code/test.txt

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File Version Author Date Message
Rmd fe41a93 brimittleman 2019-07-02 add prop of tested genes
html 2a63cde brimittleman 2019-07-01 Build site.
Rmd 8d36f9b brimittleman 2019-07-01 add res
html 5ba28ec brimittleman 2019-07-01 Build site.
Rmd 6db6003 brimittleman 2019-07-01 add qtl code
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Rmd 75b84f4 brimittleman 2019-07-01 add code premature term

library(reshape2)
library(workflowr)
This is workflowr version 1.4.0
Run ?workflowr for help getting started
library(tidyverse)
── Attaching packages ─────────────────────────────────────── tidyverse 1.2.1 ──
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✔ tidyr   0.8.3       ✔ stringr 1.3.1  
✔ readr   1.3.1       ✔ forcats 0.3.0  
── Conflicts ────────────────────────────────────────── tidyverse_conflicts() ──
✖ dplyr::filter() masks stats::filter()
✖ dplyr::lag()    masks stats::lag()

Many papers have started to talk about premature termination. Premature terminated isoforms may be truncated protein or may be degraded. I am going to create a measure for this and test for genetic variation associated with it in my data. The measure will be sum of the reads in intronic PAS and the sum of the UTR reads. I will use leafcutter to put the ratios onto a normal distribution. I will then test for QTLs these ratios.

mkdir ../data/PreTerm_pheno

Prepare phenotype

Total

gene start and end

genes=read.table("/project2/gilad/briana/genome_anotation_data/RefSeq_annotations/FullTranscriptByName.bed", col.names = c("chr", "Gene_start", "Gene_end", "gene", "score", "strand"),stringsAsFactors = F) %>% select(chr,Gene_start, Gene_end, gene)
totalPAS=read.table("../data/phenotype_5perc/APApeak_Phenotype_GeneLocAnno.Total.5perc.fc.gz",stringsAsFactors = F,header = T) 


totalPASPheno=totalPAS %>% melt(id.vars="chrom", variable.name="Ind", value.name = "ratio") %>% separate(ratio, into=c("count", "geneCount"), sep="/") %>% separate(chrom, into=c("chr", "start", "end", "geneID"), sep=":") %>% separate(geneID, into=c("gene", "loc","strand", "PAS"), sep="_") %>% filter(loc=="utr3" | loc=="intron") %>% inner_join(genes,by=c("chr", "gene"))%>% mutate(gene=paste(chr,Gene_start, Gene_end, gene,sep=":")) %>% group_by(Ind,gene,loc) %>% summarise(SumCount=sum(as.integer(count))) %>% ungroup() %>% group_by(Ind,gene) %>% mutate(nType=n()) %>% filter(nType==2) %>% spread(loc, SumCount) %>% mutate(total=intron+utr3,PreTermInt=paste(intron,total, sep="/"),PreTermUTR=paste(utr3,total, sep="/")) %>% select(-nType, -intron,-utr3,-total)


totalPASPheno_melt= totalPASPheno %>% melt(id.vars=c("Ind", "gene"), variable.name="Type", value.name = "Value") %>% mutate(chrom=paste(gene, Type, sep="_")) %>% spread(Ind, Value) %>% select(-gene, -Type)


#write.table(totalPASPheno_melt,"../data/PreTerm_pheno/Total_preterminationPheno.txt",quote=F, row.names=F,col.names=T, sep=" ")
#python2
gzip ../data/PreTerm_pheno/Total_preterminationPheno.txt
python prepare_phenotype_table.py ../data/PreTerm_pheno/Total_preterminationPheno.txt.gz

#activate env  

sh ../data/PreTerm_pheno/Total_preterminationPheno.txt.gz_prepare.sh

#top 2 pcs
head -n 3  ../data/PreTerm_pheno/Total_preterminationPheno.txt.gz.PCs > ../data/PreTerm_pheno/Total_preterminationPheno.txt.gz.2PCs 

Nuclear

nuclearPAS=read.table("../data/phenotype_5perc/APApeak_Phenotype_GeneLocAnno.Nuclear.5perc.fc.gz",stringsAsFactors = F,header = T) 


nuclearPASPheno=nuclearPAS %>% melt(id.vars="chrom", variable.name="Ind", value.name = "ratio") %>% separate(ratio, into=c("count", "geneCount"), sep="/") %>% separate(chrom, into=c("chr", "start", "end", "geneID"), sep=":") %>% separate(geneID, into=c("gene", "loc","strand", "PAS"), sep="_") %>% filter(loc=="utr3" | loc=="intron") %>% inner_join(genes,by=c("chr", "gene"))%>% mutate(gene=paste(chr,Gene_start, Gene_end, gene,sep=":")) %>% group_by(Ind,gene,loc) %>% summarise(SumCount=sum(as.integer(count))) %>% ungroup() %>% group_by(Ind,gene) %>% mutate(nType=n()) %>% filter(nType==2) %>% spread(loc, SumCount) %>% mutate(total=intron+utr3,PreTermInt=paste(intron,total, sep="/"),PreTermUTR=paste(utr3,total, sep="/")) %>% select(-nType, -intron,-utr3,-total)


nuclearPASPheno_melt= nuclearPASPheno %>% melt(id.vars=c("Ind", "gene"), variable.name="Type", value.name = "Value") %>% mutate(chrom=paste(gene, Type, sep="_")) %>% spread(Ind, Value) %>% select(-gene, -Type)


#write.table(nuclearPASPheno_melt,"../data/PreTerm_pheno/Nuclear_preterminationPheno.txt",quote=F, row.names=F,col.names=T, sep=" ")
#python2
gzip ../data/PreTerm_pheno/Nuclear_preterminationPheno.txt
python prepare_phenotype_table.py ../data/PreTerm_pheno/Nuclear_preterminationPheno.txt.gz
#env
sh ../data/PreTerm_pheno/Nuclear_preterminationPheno.txt.gz_prepare.sh

#top 2 pcs
head -n 3  ../data/PreTerm_pheno/Nuclear_preterminationPheno.txt.gz.PCs > ../data/PreTerm_pheno/Nuclear_preterminationPheno.txt.gz.2PCs 

Call QTLs

Sample list from previous work

mkdir ../data/PrematureQTLNominal
mkdir ../data/PrematureQTLPermuted
sbatch PrematureQTLNominal.sh
sbatch PrematureQTLPermuted.sh

May want to only test one number per gene but do this for now because I want to take advantage of the leafcutter normalization software.

cat ../data/PrematureQTLPermuted/Total_preterminationPheno.txt.gz.qqnorm_chr* > ../data/PrematureQTLPermuted/Total_preterminationPheno.txt.gz.qqnorm_AllChr.txt

cat ../data/PrematureQTLPermuted/Nuclear_preterminationPheno.txt.gz.qqnorm_chr* > ../data/PrematureQTLPermuted/Nuclear_preterminationPheno.txt.gz.qqnorm_AllChr.txt

Tot

totRes=read.table("../data/PrematureQTLPermuted/Total_preterminationPheno.txt.gz.qqnorm_AllChr.txt", stringsAsFactors = F,col.names = c("pid", "nvar", "shape1", "shape2", "dummy", "sid", "dist", "npval", "slope", "ppval", "bpval"))

totRes$bh=p.adjust(totRes$bpval, method="fdr")

totRes_sig=totRes %>% filter(-log10(bh)>1) 


totRes_sig_genes=totRes_sig %>% separate(pid, into=c("chr","start","end", "geneID"), sep=":") %>% separate(geneID, into=c("gene", "Frac"),sep="_") %>% select(gene) %>% unique()

write.table(totRes, file = "../data/PrematureQTLPermuted/Total_preterminationPheno.txt.gz.qqnorm_AllChrBH.txt", col.names = T, row.names = F, quote = F)
nrow(totRes_sig_genes)
[1] 40

Proportion of genes tested:

tottested_genes=totRes %>% separate(pid, into=c("chr","start","end", "geneID"), sep=":") %>% separate(geneID, into=c("gene", "Frac"),sep="_") %>% select(gene) %>% unique()
nrow(totRes_sig_genes)/nrow(tottested_genes)
[1] 0.01162453

qqplot:

qqplot(-log10(runif(nrow(totRes))), -log10(totRes$bpval),ylab="-log10 Total permuted pvalue", xlab="Uniform expectation", main="Total premature termination")
abline(0,1)

Nuclear:

nucRes=read.table("../data/PrematureQTLPermuted/Nuclear_preterminationPheno.txt.gz.qqnorm_AllChr.txt", stringsAsFactors = F,col.names = c("pid", "nvar", "shape1", "shape2", "dummy", "sid", "dist", "npval", "slope", "ppval", "bpval"))

nucRes$bh=p.adjust(nucRes$bpval, method="fdr")

nucRes_sig=nucRes %>% filter(-log10(bh)>1)


nucRes_sig_genes=nucRes_sig %>% separate(pid, into=c("chr","start","end", "geneID"), sep=":") %>% separate(geneID, into=c("gene", "Frac"),sep="_") %>% select(gene) %>% unique()


write.table(nucRes, file = "../data/PrematureQTLPermuted/Nuclear_preterminationPheno.txt.gz.qqnorm_AllChrBH.txt", col.names = T, row.names = F, quote = F)
nrow(nucRes_sig_genes)
[1] 106

Proportion of genes tested:

nuctested_genes=nucRes %>% separate(pid, into=c("chr","start","end", "geneID"), sep=":") %>% separate(geneID, into=c("gene", "Frac"),sep="_") %>% select(gene) %>% unique()
nrow(nucRes_sig_genes)/nrow(nuctested_genes)
[1] 0.02061856

qqplot:

qqplot(-log10(runif(nrow(nucRes))), -log10(nucRes$bpval),ylab="-log10 Nuclear permuted pvalue", xlab="Uniform expectation", main="Nuclear premature termination")
abline(0,1)

More likely in nuclear:

prop.test(x=c(nrow(nucRes_sig_genes),nrow(totRes_sig_genes)), n=c(nrow(nuctested_genes),nrow(tottested_genes)),alternative = "greater")

    2-sample test for equality of proportions with continuity
    correction

data:  c(nrow(nucRes_sig_genes), nrow(totRes_sig_genes)) out of c(nrow(nuctested_genes), nrow(tottested_genes))
X-squared = 9.4405, df = 1, p-value = 0.001061
alternative hypothesis: greater
95 percent confidence interval:
 0.004317405 1.000000000
sample estimates:
    prop 1     prop 2 
0.02061856 0.01162453 

sessionInfo()
R version 3.5.1 (2018-07-02)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Scientific Linux 7.4 (Nitrogen)

Matrix products: default
BLAS/LAPACK: /software/openblas-0.2.19-el7-x86_64/lib/libopenblas_haswellp-r0.2.19.so

locale:
 [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
 [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8    
 [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   
 [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 
 [9] LC_ADDRESS=C               LC_TELEPHONE=C            
[11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
 [1] forcats_0.3.0   stringr_1.3.1   dplyr_0.8.0.1   purrr_0.3.2    
 [5] readr_1.3.1     tidyr_0.8.3     tibble_2.1.1    ggplot2_3.1.1  
 [9] tidyverse_1.2.1 workflowr_1.4.0 reshape2_1.4.3 

loaded via a namespace (and not attached):
 [1] Rcpp_1.0.0       cellranger_1.1.0 compiler_3.5.1   pillar_1.3.1    
 [5] git2r_0.25.2     plyr_1.8.4       tools_3.5.1      digest_0.6.18   
 [9] lubridate_1.7.4  jsonlite_1.6     evaluate_0.12    nlme_3.1-137    
[13] gtable_0.2.0     lattice_0.20-38  pkgconfig_2.0.2  rlang_0.3.1     
[17] cli_1.0.1        rstudioapi_0.10  yaml_2.2.0       haven_1.1.2     
[21] withr_2.1.2      xml2_1.2.0       httr_1.3.1       knitr_1.20      
[25] hms_0.4.2        generics_0.0.2   fs_1.2.6         rprojroot_1.3-2 
[29] grid_3.5.1       tidyselect_0.2.5 glue_1.3.0       R6_2.3.0        
[33] readxl_1.1.0     rmarkdown_1.10   modelr_0.1.2     magrittr_1.5    
[37] whisker_0.3-2    backports_1.1.2  scales_1.0.0     htmltools_0.3.6 
[41] rvest_0.3.2      assertthat_0.2.0 colorspace_1.3-2 stringi_1.2.4   
[45] lazyeval_0.2.1   munsell_0.5.0    broom_0.5.1      crayon_1.3.4